WO2004010780A2

WO2004010780A2 - Innocuous intracellular ice

Info

Publication number: WO2004010780A2
Application number: PCT/US2003/024147
Authority: WO
Inventors: Jason Paul Acker; Locksley Earl Mcgann
Original assignee: General Biotechnology LLC
Current assignee: General Biotechnology LLC
Priority date: 2002-07-30
Filing date: 2003-07-30
Publication date: 2004-02-05
Anticipated expiration: 2005-01-30
Also published as: AU2003258001A8; WO2004010780A3; AU2003258001A1

Abstract

INNOCUOUS INTRACELLULAR ICE ABSTRACT OF THE DISCLOSURE The present invention relates to a method for cryopreserving cells and maintaining cell viability. The method comprises the steps of predetermining the nucleation temperature for extracellular and intracellular ice formation for the cells, cooling the cells to the predetermined nucleation temperature, nucleating intracellular ice formation, and cooling the cells to a temperature lower than the predetermined nucleation temperature. In another embodiment a method is provided for cryopreserving cells and maintaining cell viability without the use of cryoprotective compounds. The method comprises the step of cooling the cells under conditions to optimize controlled innocuous intracellular ice formation.

Description

INNOCUOUS INTRACELLULAR ICE

FIELD OF THE INVENTION

The invention relates to a method for cryopreserving cells while maintaining cell viability. More particularly, cells are cryopreserved while maintaining cell viability by controlling innocuous intracellular ice formation.

BACKGROUND AND SUMMARY OF THE INVENTION

The successful cryopreservation of a wide variety of cell types has been a result of the development of novel techniques to minimize cell damage.

Biophysical studies have provided a detailed understanding of the biological freezing response. During freezing, cells are dehydrated as the extracellular solutes concentrate due to the removal of pure water from the solution in the form of ice. It has been proposed that damage during slow cooling results from then excessive dehydration of the cell and the increased intracellular and extracellular solute concentrations. During rapid cooling, the formation of extracellular ice and the concentration of solutes occur too quickly for the cell to respond by exosmosis. This results in the cytoplasm becoming increasing supercooled with a concomitant increase in the probability of freezing. This is termed intracellular ice formation (IIF) and in cells and tissues it has been proposed that the formation of intracellular ice is inherently lethal. The conventional approach to cryopreservation has therefore been to balance the toxic effects of high concentrations of solutes using chemical cryoprotectants with cooling rates slow enough to avoid ILF, but rapid enough to minimize cryoprotectant exposure. The application of classic freezing techniques to the cryopreservation of cells, tissues and tissue models has assumed that intracellular ice is lethal. As it has been shown that intracellular ice formation in cells in suspension occurs during rapid freezing and that rapid freezing causes cell death, it has been assumed that IIF causes cell death. However, there is no direct evidence in the literature to lead one to conclude that the mere presence of ice within a cell is lethal. Early studies with rabbit corneal tissue, skin, tumors and plant tissue could not correlate the presence of intracellular ice with cell death. While avoiding intracellular ice has been a common practice in the cryopreservation of cell suspensions, recent studies with tissue model systems suggest that this may be much more difficult to achieve in tissues. It has been demonstrated in cultured hepatocytes, fibroblasts and keratinocytes that there is a significant increase in the formation of intracellular ice at lower cooling rates and at higher subzero temperatures than occurs in the same cells in suspension. It has been further reported that cell-cell contact can facilitate the nucleation of ice between adjacent cells increasing the incidence of ILF in tissue models. Preventing the formation of ILF using traditional cryopreservation protocols may not be possible using current freezing technology. However, recent reports examining ILF in tissue models coupled with the growing evidence that innocuous ILF may be an adaptive mechanism used by insect species that survive freezing, suggests that the mechanism by which intracellular ice forms in tissue models and intact organisms has a greater impact on cell viability than the mere presence of intracellular ice. In one embodiment a method is provided for cryopreserving cells and maintaining cell viability. The method comprises the steps of predetermining the nucleation temperature for extracellular and intracellular ice formation for the cells, cooling the cells to the predetermined nucleation temperature, nucleating intracellular ice formation, and cooling the cells to a temperature lower than the predetermined nucleation temperature.

In this embodiment the predetermined nucleation temperature can be from about -3°C to about -40°C, from about -4°C to about -40°C, from about -5°C to about -40°C, from about -5°C to about -30°C, from about -5°C to about -20°C, or from about -5°C to about -10°C. Alternatively, other temperature ranges disclosed herein can be used.

In another embodiment the cells are cooled to about -30°C to about -200°C, about -30°C to about -100°C, or to about -40°C following nucleation of intracellular ice formation. Alternatively, other temperature ranges disclosed herein can be used. In still another embodiment intracellular ice formation is nucleated by a mechanism selected from the group consisting of osmotic cycling, thermal shock, chemical permeabilization, electroporation, and surface-catalyzed nucleation. In yet another embodiment a method is provided for cryopreserving cells and maintaining cell viability without the use of cryoprotective compounds. The method comprises the step of cooling the cells under conditions to optimize controlled innocuous intracellular ice formation.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the cumulative incidence of intracellular ice formation in V-79W Chinese hamster fibroblasts (A) and Madin Darby canine kidney epithelial cells (B). Mean ± SEM (n = 6). Fig. 2 shows the effect of intracellular ice formation on the post-thaw survival of V-79W and MDCK single attached cells. The incidence of intracellular ice (left column in each group), membrane integrity (second column from left in each group), metabolic activity (third column from the left in each group), and clonogenic function (right column in each group) is plotted as a function of nucleation temperature. Mean ± SEM (n = 6).

Fig. 3 shows the effect of intracellular ice formation on the post-thaw survival of V-79W and MDCK confluent cell monolayers. The incidence of intracellular ice (left column in each group), membrane integrity (second column from left in each group), metabolic activity (third column from the left in each group), and clonogenic function (right column in each group) is plotted as a function of nucleation temperature. Mean ± SEM (n - 6).

Fig. 4 shows the metabolic activity of V-79W and MDCK single attached cells and confluent monolayers and confluent monolayers following freezing using three different cryopreservation protocols. The survival of cells frozen using a standard freezing protocol (left column in each group), a standard freezing protocol + DMSO (middle column in each group), or a modified freezing protocol involving intracellular ice formation (right column in each group) is shown. Mean ± SEM (n = 6).

Fig. 5 shows the incidence of intracellular ice formation in V-79W and MDCK single attached cells and confluent monolayers following nucleation at -5°C (left bar in each group) or -10°C (right bar in each group). Mean ± SEM (n = 6). The asterisk denote a statistically significant difference (p < 0.05; Student's t test) in the incidence of IIF at the two different nucleation temperatures. Figs. 6 A and B show the responses of V-79W fibroblasts (panels A and B) and MDCK epithelial cells (panels C and D) to graded freezing. The samples were assessed for membrane integrity (panels A and C) and metabolic activity (panels B and D) following nucleation of extracellular ice. Single attached cells nucleated at -5°C (open circle, solid line) or -10°C (closed circle, dashed line) and confluent monolayers nucleated at -5°C (open square, solid line) or -10°C (closed square, solid line) are shown. Mean ± SEM (n = 6).

DETAILED DESCRIPTION In one embodiment a method is provided for cryopreserving cells and maintaining cell viability. The method comprises the steps of predetermining the nucleation temperature for extracellular and intracellular ice formation for the cells, cooling the cells to the predetermined nucleation temperature, nucleating intracellular ice formation, and cooling the cells to a temperature lower than the predetermined nucleation temperature. In another embodiment a method is provided for cryopreserving cells and maintaining cell viability without the use of cryoprotective compounds. The method comprises the step of cooling the cells under conditions to optimize controlled innocuous intracellular ice formation.

Any cell type, cell line, tissue, organ, or whole organism known in the art can be cryopreserved using the presently disclosed method. The cell types or cell lines for use in the presently described method include, but are not limited to, animal cells, plant cells, insect cells, mammalian cells, and human cells. Transgenic cells, genetically engineered cells, or transformed cells can be used and the cells can be nucleated or non-nucleated. The cell types or cell lines can comprise anchorage- dependent cells or anchorage-independent cells (e.g., cells grown in suspension) and the cells can be differentiated or undifferentiated. The cells can be cultured cell types or cell lines or the cells can be derived from native tissues, organs, whole organisms, or the cells can be derived from engineered cells, tissues, organs, or whole organisms. If cultured, the cell types or cell lines can be grown in suspension or as monolayers and the cell types or cell lines can be subconfluent or confluent.

Cells cultured as either anchorage-dependent or anchorage- independent cell types or cell lines are known to those skilled in the art to include, but are not limited to, tumor cells, hematopoietic cells, fibroblasts, hepatocytes, epithelial cells, keratinocytes, stem cells, smooth muscle cells, dendritic cells or other immune cells such as macrophages, T-cells, B-cells, or mast cells, stromal cells, chondrocytes, endothelial cells, mesenchymal cells, or any other cell type or cell line known in the art that can be cultured. Alternatively, the cells can be any cell type from any tissue or organ source including, but not limited to, blood, lung, heart, kidney, stroma, brain, liver, muscle, skin, ovaries, and testes. Cells useful in the disclosed method can be comprised of a single cell type or cell line or of multiple cell types or cell lines.

For the method of the present invention, the nucleation temperature is determined for extracellular and or intracellular ice formation for the specific cell type or cell line to be cryopreserved. The nucleation temperature for extracellular and/or intracellular ice formation can be determined using any technique known to those skilled in the art such as the standard flash method disclosed herein where the presence of intracellular ice is denoted by a visible darkening of the cytoplasm due to the scattering of light by intracellular ice crystals. Alternatively, any fluorometric technique can be used, such as a technique using a nucleic acid-binding fluorescent stain (e.g., SYTO 13; Molecular Probes, Eugene, OR), as described herein, to directly assess the incidence of intracellular ice formation. Any other technique known to the skilled artisan to be useful for determining the nucleation temperature for extracellular and/or intracellular ice formation can be used. In alternative embodiments the predetermined nucleation temperature can be from about 0 °C to about -40°C, from about -2°C to about -40°C, from about -3°C to about -40°C, from about -4°C to about -40°C, from about -5°C to about -40°C, from about -5°C to about -30°C, from about -5°C to about -20°C, from about -5°C to about -15°C, from about -5°C to about -13°C, from about -5°C to about -12°C, or from about -5°C to about -10°C.

In accordance with the invention, the cell type or cell line to be cryopreserved is cooled at any rate to the predetermined nucleation temperature, and intracellular ice formation is induced. In one embodiment the cell type or cell line to be cryopreserved is cooled to a temperature higher than the predetermined nucleation temperature (e.g., 0°C) prior to cooling the cell type or cell line to the predetermined nucleation temperature. Intracelluar ice formation is induced when the cell type or cell line is cooled to the predetermined nucleation temperature. hi accordance with the invention, nucleation of intracellular ice formation means that an active step is performed to induce intracellular ice formation. In alternative embodiments, for example, intracellular ice formation can be induced (i.e., nucleated) by a method selected from the group consisting of osmotic cycling, thermal shock, chemical permeabilization, electroporation, and surface-catalyzed nucleation (e.g., using forceps to induce intracellular ice formation). Alternatively, any other method known to those skilled in the art for inducing intracellular ice formation can be used.

In one embodiment the cell type or cell line to be cryopreserved is cryopreserved in the absence of cryoprotectants. Cryoprotectants that are known in the art are small molecular weight compounds, such as dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, and the like.

Upon nucleation of intracellular ice formation, intracellular ice is formed within the cells, and the percentage of cells wherein intracellular ice is formed can be greater than 20%o of the cells, greater than 30% of the cells, greater than 40% of the cells, greater than 45% of the cells, greater than 50% of the cells, greater than 55%ι of the cells, greater than 60% of the cells, greater than 65% of the cells, greater than 70%) of the cells, greater than 75% of the cells, greater than 80% of the cells, greater than 85% of the cells, greater than 90% of the cells, or greater than 95% of the cells.

In accordance with the disclosed method, conditions to optimize controlled innocuous intracellular ice formation include those conditions described herein such as cryopreserving cell types, including cell types of tissues, organs, or whole organisms, or cell lines, that are confluent, and/or inducing intracellular ice formation at a temperature that causes intracellular ice to be formed in a high percentage of the cells (e.g., greater than 60% of the cells, greater than 65% of the cells, greater than 70% of the cells, greater than 75% of the cells, greater than 80% of the cells, greater than 85%> of the cells, greater than 90% of the cells, or greater than 95% of the cells). After nucleation of intracellular ice formation, the cell type or cell line to be cryopreserved can be cooled to a predetermined storage temperature, lower than the predetermined nucleation temperature. In alternative embodiments the cell type or cell line can be cooled for storage to about -30 °C to about -200 °C, about -30°C to about -150°C, about -30°C to about -100°C, about -30°C to about -80°C, about -30°C to about -60°C, about -30°C to about -40°C, or to about -40°C following nucleation of intracellular ice formation. The rate of cooling can be from about l°C/min to about 500°C/min (i.e., slow or rapid cooling). The cell type or cell line can be thawed, when needed, and the rate of thawing can be from about l°C/min to about 500°C/min (i.e., slow or rapid thawing).

Cell types or cell lines that are cryopreserved using the method disclosed herein maintain cell viability. In alternative embodiments, the percentage of cells that maintain viability can be greater than 20%> of the cells, greater than 30% of the cells, greater than 40%) of the cells, greater than 45% of the cells, greater than 50% of the cells, greater than 55%> of the cells, greater than 60% of the cells, greater than 65%> of the cells, greater than 70% of the cells, greater than 75% of the cells, greater than 80%) of the cells, greater than 85% of the cells, greater than 90% of the cells, or greater than 95%> of the cells. Cell viability can be assessed as described herein. For example, cell viability can be assessed by examining membrane integrity, metabolic activity, or clonogenic function as described below.

The following examples are illustrative of the presently claimed method and are not intended to limit the invention. Variations and modifications of the exemplified method obvious to one skilled in the art are also intended to be within the scope of the invention as specified in the claims.

EXAMPLES Cell culture

Two cell lines were used to investigate the effect of intracellular ice formation on cell viability. The first was the Madin Darby Canine Kidney (MDCK; CCL34 ATCC) epithelial cell line. These cells were incubated at 37 °C in an atmosphere of 95% air + 5%> carbon dioxide in minimum essential media (MEM) supplemented with 10% v/v fetal bovine serum (all components from GLBCO Laboratories, Grand Island, NY). Cells were grown in tissue culture flasks (25 cm²; Corning Glass Works, Corning, NY) and harvested by exposure to a 0.25% trypsin- EDTA solution (GLBCO) for 10 min at 37 °C. The MDCK cells were resuspended in supplemented MEM to obtain cell suspensions and then plated on sterilized cover

4 slips (12 mm circle, Fisher Brand) at a concentration of 2x10 cells/mL. The cover slips were kept in petri dishes in an incubator for 12 hours to allow the cells to attach and 3 days to allow the growth of a confluent monolayer.

The second cell line was the V-79W line of Chinese hamster fibroblasts. Cells were incubated at 37 °C in an atmosphere of 95% air + 5% carbon dioxide in minimum essential medium (MEM) with Hanks' salts, 16 mmol L sodium bicarbonate, 2 mmol/L L-glutamine and 10%> fetal bovine serum supplemented with antibiotics (penicillin G (50 units/mL), streptomycin (50 μg/mL)) (all components from GLBCO Laboratories, Grand Island, NY). Cells were grown in tissue culture flasks (25 cm²; Corning Glass Works) and harvested by exposure to a 0.25% trypsin solution (GD3CO) for 10 min at 37°C. The fibroblasts were resuspended in supplemented MEM. Sterilized cover slips (12 mm circle, FISHER Brand) were placed in a petri dish (FISHER Brand, 100 x 15 mm) and covered with 15 mL of

5 supplemented MEM containing 3x10 cells. The petri dishes were incubated for 12 hours to allow the cells to attach and 3 days to allow the growth of a confluent monolayer.

Cryomicroscope and video system

The cryomicroscope and video system used for this study is known in the art. Briefly, it consisted of a Zeiss fluorescent microscope (Carl Zeiss, Germany), a CCD video camera (ZVS-47DEC, Carl Zeiss), a video recorder (GX4, Panasonic, Japan) and a convection cryostage. The cryostage was connected to a computer- controlled interface (Great Canadian Computer Company, Spruce Grove, Canada). The computer monitored the temperature by analyzing the voltage from a thermocouple on the stage and via a proportional controller circuit; heat was added as necessary to allow the stage to follow a user defined thermal protocol.

Membrane integrity assay

A dual fluorescent staining technique was used for the quantitative assessment of the integrity of the cell plasma membrane. SYTO 13, a permeant live cell nucleic acid dye (1.25 μM) and ethidium bromide (EB; Sigma Chemical

Company, Mississuaga, ON; 2.5 μM) where used to differentially stain the cells. Percent survival based on membrane integrity was calculated as the number of SYTO positive cells over the total number of cells (SYTO and EB positive) using the following equation:

. , total SYTO positive cells . ..

% survival = x 100 total SYTO positive + total EB positive cells

Metabolic activity assay

AlamarBlue™ (Biosource International, CA) was used to assess the overall metabolic activity of the confluent monolayers and single attached cells post- thaw. AlamarBlue was added to tissue culture media (5-10% v/v) and confluent monolayers (MDCK and V-79W) were incubated in this solution for 12 hours at 37°C. Single attached cells (MDCK and V-79W) were incubated for 24 hours at 37°C in 10%) alamarBlue. An aliquot (100 μL) of the media was removed and measured on a spectrophotometer (570-600 nm; UVmax, Molecular Dynamics, CA). Percent survival based on metabolic activity was calculated as the mean percent difference in reduction between the experimental samples and the controls using the following equations:

• . A_τw - (AT_™ x R_n) for test well . _{- Λ}

% survival = ^LW , " — ^ x 100

A_LW - A_jpy xR unfrozen control

where A_LW is the absorbance value of the sample minus the absorbance of the media only at 570 nm, A_HW is the absorbance value of the sample minus the absorbance of the media only at 600 nm. R given by:

AO

R„ LW

AO_j^_r where AO_T L ,W-, is the absorbance of alamarBlue in media minus the absorbance of media only at 570 and AO _w is the absorbance of alamarBlue in media minus the absorbance of media only at 600 nm.

Clonogenic assessment

The cells were collected from the coverslips by exposure to a 0.25%ι trypsin-EDTA solution and diluted to 750 cells/mL. Tissue culture flasks were seeded with 150 cells/flask and incubated at 37 °C for 5 days. The tissue culture media was removed and the colonies were fixed with 70% isopropanol, stained with trypan blue, and rinsed with distilled water before being counted. Percent survival based on clonogenic function was calculated as the mean of the experimental colony counts expressed as a percentage of the mean unfrozen controls.

Intracellular ice formation assay

The formation of intracellular ice was detected using the standard flash method known in the art where the presence of intracellular ice is denoted by a visible darkening of the cytoplasm due to the scattering of light by intracellular ice crystals. hi addition to the flash method, a fluorometric technique was used to assist in the identification of ILF in the MDCK and V-79W confluent monolayers.

For the fluorometric technique, a nucleic acid-binding fluorescent stain (SYTO 13; Molecular Probes, Eugene, OR) was used to directly assess the incidence of ILF in the V-79W and MDCK single attached cells and confluent monolayers following extracellular ice nucleation. Prior studies have shown that the formation of intracellular ice disrupts the structures stained by fluorescent dyes resulting in a distinctive "honeycomb" pattern that can be used to directly quantify the incidence of ILF. Samples were stained with 12.5μM SYTO prior to being placed into a pre-cooled alcohol bath. After equilibration at the pre-set temperature (-5 or -10°C) ice was nucleated in the samples with cold forceps and then held at the subzero temperature for 5 min before being transferred to an inverted fluorescent microscope and immediately assessed. Images were acquired before the samples could completely melt. The incidence of intracellular ice formation was determined using this fluorometric method and calculated as the percent of cells that form intracellular ice compared to the total cells in the sample.

Complete viability assessment procedure

Madin-Darby canine kidney (MDCK) epithelial cells and V-79W hamster fibroblasts were either attached individually or grown to confluency on glass coverslips, then stained with SYTO and EB prior to freezing. The samples were then supercooled to a defined subzero experimental temperature on a convection cryostage by cooling at 25 °C/min. The subzero experimental temperatures were carefully chosen to ensure that approximately 100% of the cells would form intracellular ice (see Fig. 1). Under video surveillance, ice was nucleated using a cold copper probe at the constant temperature and the incidence of intracellular ice formation was observed after holding for 5 min. Cells were then warmed at 25°C/min and the integrity of the cell plasma membrane was quantitatively assessed. The samples were then transferred from the cryomicroscope and assayed for metabolic function using the reduction-oxidation indicator alamarBlue. The cells were trypsinized, plated, incubated for 5 days, and the number of colony forming units was determined. The cumulative incidence of cells with intracellular ice formation was determined as a function of nucleation temperature and correlated with post-thaw survival.

Standard Freezing Protocol

Madin-Darby canine kidney epithelial cells and V-79W hamster fibroblasts were either used as single attached cells or grown to confluency on glass coverslips. The cover slips containing a confluent monolayer were placed in 15 x 45 mm glass tubes (Kimble Glass Inc.) and placed on ice for 5 min. Following this incubation at 0°C, the samples were immersed in an alcohol bath at -5 °C and allowed to cool for 5 min. Extracellular ice formation was induced in the samples using cold forceps. The bath was then cooled at 1 °C/min to -40°C where the samples were held for 5 min. All samples were then rapidly warmed in a 37°C water bath and incubated in a 10%> alamarBlue solution at 37°C. Metabolic activity was then assessed.

Standard Freezing Protocol With Cryoprotectant

MDCK and V-79W single attached cells and confluent monolayers were subjected to the standard freezing protocol with addition of 10% v/v DMSO (Cryoserv, Tera Pharmaceuticals Inc., Buena Park, CA). The cover slips containing the cells were placed in glass tubes incubated for 5 min in an ice bath and then 500 μL of a 10%) v/v DMSO solution (in supplemented tissue culture media) was added to the glass tubes. The single attached cells and confluent monolayers were allowed to incubate with the DMSO for 20 min and then 450 μL of the solution was removed prior to freezing. The samples were immersed in an alcohol bath and the above freezing protocol was used. All samples were then rapidly warmed in a 37 °C water bath, washed twice with tissue culture media to remove the cryoprotectant and incubated in a 10% alamarBlue solution at 37°C. Metabolic activity was then assessed. Unfrozen samples without DMSO served as positive growth controls.

Cryoprotective Intracellular Freezing Protocol MDCK and V-79W cells were either used as single attached cells or grown to confluency on glass coverslips. The cover slips containing single attached cells and confluent monolayers were placed in 15 x 45 mm glass tubes and placed on ice for 5 min. Following this incubation at 0°C, the samples were immersed in an alcohol bath at -10°C and allowed to cool for 5 min. Extracellular ice formation was induced at -10°C in the samples using cold forceps. At -10°C, 100% intracellular ice formation has been shown to occur in all of the V-79W and MDCK single attached cells and confluent monolayers (see Fig. 1). The bath was then cooled at l°C/min to -40°C where the samples were held for 5 min. All samples were then rapidly warmed in a 37°C water bath, incubated in a 10%> alamarBlue solution at 37°C and assessed. Another controlled-rate freezing/thawing protocol (graded freezing) was used to progressively to simulate cell injury during freezing (used only in the assay shown in Figs. 6 A and B). Confluent monolayers and single attached cells cultured on sterile glass coverslips (12 mm, Fisher scientific), were placed in glass tubes (15 x 45 mm, Kimble Glass), covered with 50 μL of tissue culture media, and immersed in an alcohol bath (Multi-Cool, FTS Systems, Stone Ridge, NY) pre-set at -5°C. Extracellular ice formation was induced in the samples using cold forceps, and the samples were held at -5°C for 5 min for the dissipation of the latent heat of fusion. The bath was then cooled at l°C/min, and at different subzero temperatures (-5, -10, -15, -20, -30, and -40°C), samples were thawed directly in a circulating water bath at 37°C and assessed. Alternatively, extracellular ice nucleation was initiated at -10°C by placing samples into the -5°C alcohol bath for 5 min before being cooled at l°C/min to -10°C. Nucleation was induced using cold forceps, and the samples were allowed to equilibrate at -10°C for 5 min before continuing with the graded freezing protocol. Control samples cooled to and thawed from the two nucleation temperatures (-5 and -10°C) followed the above thermal profile but did not undergo extracellular ice nucleation. Statistical Analysis

Unless otherwise indicated, a one-way ANOVA test was used with a level of significance set at 0.05.

Results

As shown in Fig. 1 the incidence of intracellular ice formation in V- 79W and MDCK cells is strongly dependent on the presence of cell-cell and cell- surface adhesions and the temperature of nucleation. For both V-79W and MDCK confluent monolayers, there is a significant increase in the incidence of intracellular ice formation when compared to cell suspensions and single attached cells. In some of the experiments described below, nucleation temperatures were chosen to ensure that 100%) of the cells would form intracellular ice upon extracellular ice nucleation regardless of cell type or morphological configuration.

The incidence of intracellular ice formation, membrane integrity, metabolic activity and clonogenic function at different nucleation temperatures for V- 79W and MDCK single attached cells are shown in Fig. 2. At the experimental temperatures chosen (see x-axis of Fig. 2), approximately 100% of the cells fonned intracellular ice in both of the cell lines examined. Immediately post-thaw, there was a significant reduction in the number of cells with intact plasma membranes. Reduction in metabolic activity assessed using alamarBlue followed a similar pattern. Incubation at 37 °C in 5% alamarBlue revealed that the V-79W and MDCK single attached cells displayed a significantly lower level of metabolic activity compared to unfrozen controls (p < 0.05).

After the evaluation of metabolic activity, single attached cells were examined for clonogenic function using a colony-forming assay. Following trypsinization, plating and incubation for 5 days, both V-79W and MDCK cells displayed no clonogenic function following intracellular ice formation at the nucleation temperatures shown. As the post-thaw incubation period would permit the repair of any sublethal freeze-thaw damage, the absence of clonogenic function indicates that ILF rrreversibly damaged cellular processes in these single attached cells. Statistical analysis revealed that there was no difference between the survival based on membrane integrity, metabolic activity or clonogenic function at each experimental temperature for V-79W and MDCK single attached cells (p<0.05, Tukey's multiple comparison test). Thus, following intracellular ice formation there was a significant reduction in the three indicators of cell viability for single attached cells. These data demonstrate that ILF is detrimental in V-79W and MDCK single attached cells. V-79W and MDCK confluent cell monolayers were subjected to low temperature conditions where ILF was shown to occur and then evaluated using the three different assessment techniques. The effect of ILF on the post-thaw survival of V-79W and MDCK confluent monolayers is shown in Fig. 3. As MDCK monolayers have been shown to form intracellular ice at high subzero temperatures (see Fig. 1), it was possible to extend the range of nucleation temperatures examined for MDCK cells. At the experimental temperatures chosen, approximately 100% of the cells formed intracellular ice in both V-79W and MDCK monolayers. Post-thaw assessment of membrane integrity revealed that the majority of the cells of both V- 79W and MDCK monolayers had intact plasma membranes, were metabolically active and were able to form colonies when cultured (see Fig. 3). There was no statistical difference in the survival indices across nucleation temperatures for V-79W and MDCK monolayers. Approximately 80%» of the cells from V-79W and MDCK monolayers were viable following ILF indicating that intracellular freezing does not result in significant damage to V-79W or MDCK confluent monolayers. Thus, the presence of intracellular ice in V-79W and MDCK confluent cell monolayers is innocuous.

The metabolic activity of V-79W and MDCK single attached cells and confluent monolayers following freezing using the three freezing protocols described above is shown in Fig. 4. Nucleation at -5°C and cooling at 1 °C/min did not result in a high degree of cell recovery for single attached cells. The recovery of metabolic activity following freezing using the standard freezing protocol described above was 15.0 ± 4.9% for V-79W cells and 5.4 ± 2.4 % for MDCK cells. This is consistent with the results observed in Fig. 2. Freezing after equilibration with the cryoprotectant dimethyl sulfoxide (DMSO) resulted in an increase in the metabolic activity of V-79W and MDCK single attached cells. The recovery following the addition of 10% DMSO was 80.0 ± 10.7% for V-79W single attached cells and 98.5 ± 3.3% for MDCK single attached cells. This is statistically greater than the results obtained using the standard freezing protocol (V-79W_jp<0.05; MDCK/ 0.05). The addition and removal of 10% DMSO to the single attached cells in the absence of freezing did not affect cell viability (data not shown). Nucleating at a lower temperature (-10°C) to produce a higher incidence of intracellular ice resulted in a decrease in the recovery of metabolic activity in both V-79W and MDCK single attached cells. The recovery following nucleation at -10°C and cooling at l°C/min was 1.1 ± 0.6 % for V-79W and 1.9 ± 1.0 % for MDCK single attached cells. Both values are less than the results from the standard freezing protocol (V-79W =0.018; MDCK_jp=0.208) and the standard freezing protocol with DMSO (V-19W p<0. 5; MDCK / θ.05). These data show that ILF in single attached cells causes significant cell damage resulting in a negative post-thaw survival outcome.

The effect of freezing using the three different freezing protocols described above for V-79W and MDCK confluent monolayers is also shown in Fig. 4. Using the standard freezing protocol of nucleating at -5°C and cooling at l°C/min resulted in a degree of post-thaw cell recovery similar to single attached cells. The metabolic activity of confluent monolayers following freezing using the standard protocol was 22.5 ± 9.8% for V-79W and 19.9 ± 10.5 % for MDCK. The addition of a cryoprotectant significantly enhanced the recovery of metabolic activity for V-79W and MDCK confluent monolayers. The recovery following the addition of 10%> DMSO was 90.5 ± 10.7 % for V-79W confluent cells and 84.6 ± 8.4% for MDCK confluent cells. This is statistically greater than the results obtained using the standard freezing protocol (V-79W ?<0.05; MDCK/ θ.05). In the absence of freezing, the addition and removal of 10%) DMSO to the confluent monolayers did not affect cell viability (data not shown).

By lowering the nucleation temperature from -5°C to -10°C, formation of intracellular ice was achieved in all of the constituent cells in the confluent monolayers (see Fig. 1). By inducing complete intracellular ice formation, a higher degree of cell recovery was obtained following slow cooling and rapid thawing. The recovery of metabolic activity was 40.4 ± 3.7 % for V-79W and 58.2 ± 8.4 % for MDCK confluent monolayers. These values are greater than the results obtained using the standard freezing protocol (V-79Wp=0.118; MDCKp=0.Ql7). Thus, intracellular ice formation in confluent monolayers provides protection against the damaging effects of freezing and thawing, and inducing intracellular ice formation is an effective method for the cryopreservation of confluent cell monolayers. Ln the absence of any chemical cryoprotectant, a significant degree of cell recovery was obtained following the formation of intracellular ice.

The results presented in Fig. 5 further demonstrate that ILF prevents damage against freezing and thawing. To modulate the degree of intracellular ice formation in single attached cells and confluent monolayers, the temperature of extracellular ice nucleation was decreased from -5 to -10°C. Nucleation at -10°C resulted in a significant increase in the formation of intracellular ice in V-79W and MDCK single attached cells and V-79W confluent monolayers compared to nucleation at -5°C (Fig. 5). The post-thaw metabolic activity and membrane integrity of V-79W and MDCK single attached cells and confluent monolayers following nucleation (-5 and -10 °C) and graded freezing is shown in Fig. 6 A and B. For V-79W single attached cells, decreasing the extracellular ice nucleation temperature from -5 to -10 °C increased the incidence of intracellular ice from ~0 to > 90% (Fig. 5). The membrane integrity (Fig. 6 panel A) and metabolic activity (Fig. 6 panel B) of V-79W single attached cells that were rapidly thawed from -5°C were significantly greater (p < 0.05; ANOVA; open circles) than what was found for cells thawed from -10 °C (closed circles). A further decrease in membrane integrity and metabolic activity occurred following nucleation when the samples were cooled at l°C/min to -40°C. The increased incidence of intracellular ice formation following extracellular ice nucleation at -10 °C conesponded directly with a significant decrease in the post-thaw survival of V-79W single attached cells. Similarly, decreasing the extracellular ice nucleation temperature in MDCK single attached cells resulted in an increase in the incidence of ILF from ~60 to 100% (Fig. 5) and a decrease in both membrane integrity (in Fig 6 panel C, -5 °C = open circle and -10 °C = closed circles) and metabolic activity (Fig. 6 panel D, p<0.05, ANOVA; in Fig. 6 panel D, -5°C = open circles and - 10°C = closed circles).

In confluent monolayers of V-79W cells, reducing the extracellular ice nucleation temperature had an opposite effect on the post-thaw membrane integrity (Fig. 6 panel A) and metabolic activity (Fig. 6 panel B). Extracellular ice nucleation at -10°C (100% ILF; closed square) resulted in higher post-thaw survival than extracellular ice nucleation at -5°C (-10% ILF; open square) at all experimental temperatures down to -40°C. Ln monolayers of MDCK cells, extracellular ice nucleation at either -5 or 10°C resulted in 100% ILF, with correspondingly high recovery on subsequent slow cooling (in Fig. 6 panels C and D, -5°C = open squares and -10°C = closed squares). Notably, cell survival in confluent monolayers of both cell types after ice nucleation at -10°C is remarkably high, particularly since no chemical cryoprotectant was added. Cell survival in the monolayer was highest when the incidence of ILF was close to 100%).

The concept of inducing the formation of intracellular ice as a means of affording cryoprotection to tissue model systems is contrary to popular thinking. The results presented here show that induced intracellular ice formation is an effective alternative to classic cryopreservation techniques.

Claims

CLALMS:

1. A method for cryopreserving cells and maintaining cell viability, the method comprising the steps of: predetermining the nucleation temperature for extracellular and intracellular ice formation for the cells; cooling the cells to the predetermined nucleation temperature; nucleating intracellular ice formation; and cooling the cells to a temperature lower than the predetermined nucleation temperature.

2. The method of claim 1 wherein the cells are confluent.

3. The method of claim 1 wherein the cells are of non-human animal origin.

4. The method of claim 1 wherein the cells are of human origin.

5. The method of claim 1 wherein the cells are cell types selected from the group consisting of tissues, organs, and whole organisms of non-human animal origin.

6. The method of claim 1 wherein the cells are components of cell types selected from the group consisting of tissues, organs, and whole organisms of human origin.

7. The method of claim 1 wherein the predetermined nucleation temperature is from about -3 °C to about -40 °C.

8. The method of claim 1 wherein the predetermined nucleation temperature is from about -4 °C to about -40 °C.

9. The method of claim 1 wherein the predetermined nucleation temperature is from about -5 °C to about -40 °C.

10. The method of claim 1 wherein the predetermined nucleation temperature is from about -5 °C to about -30 °C.

11. The method of claim 1 wherein the predetermined nucleation temperature is from about -5 °C to about -20 °C.

12. The method of claim 1 wherein the predetermined nucleation temperature is from about -5°C to about -10°C.

13. The method of claim 1 wherein the cells are cooled to about -30°C to about -200°C following nucleation of intracellular ice formation.

14. The method of claim 1 wherein the cells are cooled to about -30°C to about -100°C following nucleation of intracellular ice formation.

15. The method of claim 1 wherein the cells are cooled to about -

40°C following nucleation of intracellular ice formation.

16. The method of claim 1 wherein intracellular ice formation is nucleated by a mechanism selected from the group consisting of osmotic cycling, thermal shock, chemical permeabihzation, elecfroporation, and surface-catalyzed nucleation.

17. The method of claim 1 wherein the cells are cryopreserved without the use of cryoprotective compounds.

18. The method of claim 1 wherein the cells are cooled to 0°C before being cooled to the predetermined nucleation temperature.

19. The method of claim 1 wherein intracellular ice is formed in greater than 80% of the cells upon nucleation.

20. A method for cryopreserving cells and maintaining cell viability without the use of cryoprotective compounds, said method comprising the step of cooling said cells under conditions to optimize controlled innocuous intracellular ice formation.